Glycoprotein Ib/IX/V Binding to the Membrane Skeleton Maintains Shear-Induced Platelet Aggregation

Abstract

The extracellular domain of glycoprotein (Gp) Ibα serves as the von Willebrand factor (vWf) receptor that triggers shear stress-dependent platelet aggregation. Its intracellular domain associates with actin-binding protein-280 (filamin 1a) that binds directly to filamentous actin, thereby linking the membrane skeleton to GpIbα. We examined the functional significance of GpIbα interactions with actin during platelet aggregation in response to 120 dyn/cm 2 shear stress. Lysates of resting and sheared platelets were centrifuged at ∼13,000× g for 15 min, and GpIbα was immunoprecipitated from the lysate supernatant. GpIbα and coimmunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies specific for GpIbα and actin. We observed a significant increase in the amounts of actin coimmunoprecipitating with GpIbα as platelets aggregated in response to shear stress. Actin/GpIbα interactions reached a maximum after 90 s of shear stress. Monoclonal antibody (mAb) blockade of vWf binding to GpIbα inhibited shear stress-induced platelet aggregation and actin associating with GpIbα. Pretreatment of platelets with cytochalasin D resulted in the inhibition of actin binding to GpIbα in sheared platelets and in an increase in the rate and magnitude of platelet disaggregation. These data indicate that shear stress causes changes in the association between GpIbα and the actin-based membrane skeleton. The increased interaction between GpIbα and the actin-based membrane skeleton results from shear-induced vWf binding to GpIbα and is mechanoprotective in that it maintains shear-induced aggregation of activated platelets.